This project is concerned with the mechanism of action of thymidylate synthetase (deoxyuridylate plus 5,10-methylene tetrahydrofolate yields thymidylate plus 7, 8-dihydrofolate) from amethopterin-resistant Lactobacillus casei. The problems to be investigated include (a) the importance of sulfhydryl groups in the activation and inactivation of the enzyme, (b) relationship of the carboxyl terminus to activity and tertiary structure, (c) the formation, stability, and structure of inhibitor-thymidylate synthetase complexes, (d) investigation of the role of histidine in thymidylate synthetase, (e) complementary studies with dihydrofolate reductase, (f) interactions of folates with thymidylate synthetase, (g) denaturation-renaturation studies, (h) application of circular dichroism spectroscopy to thymidylate synthetase, and (i) cellular studies to determine the effect of amethopterin resistance on the nature and levels of thymidylate synthetase and selected enzymes.